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Mineralocorticoid receptor overexpression facilitates differentiation and
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promotes survival of embryonic stem cell-derived neurons
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Abbreviated title: Mineralocorticoid receptor as neuroprotective factor
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Mathilde Munier1,2, Frédéric Law1, Geri Meduri1,3, Damien Le Menuet1,2*, and Marc Lombès1,2,4*
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* These authors contributed equally
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Authors’ information:
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1
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2
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France;
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Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre F-94275, France;
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Hôpital de Bicêtre, Le Kremlin-Bicêtre, F-94275, France.
Inserm, U693, Le Kremlin-Bicêtre, F-94276, France;
Univ Paris-Sud 11, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin-Bicêtre, F-94276,
Service de Génétique Moléculaire, Pharmacogénétique, Hormonologie, Le Kremlin-Bicêtre
Service d’Endocrinologie et Maladies de la Reproduction, Assistance Publique-Hôpitaux de Paris,
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Corresponding author’s address: Marc Lombès, INSERM U693, Faculté de Médecine Paris-Sud 11,
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63, rue Gabriel Péri, 94276 Le Kremlin-Bicêtre Cedex France. E-mail: [email protected]. Tel :
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+33 1 49 59 67 09. Fax : + 33 1 49 59 67 32
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Keywords: neuronal differentiation, mineralocorticoid receptor, apoptosis, embryonic stem cells
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This work was supported by fundings from Institut National de la Santé et de la Recherche Médicale
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(Inserm) and the Université Paris-Sud 11. MM was recipient of fellowships from the Ministère de
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l’Enseignement Supérieur et de la Recherche and the Société Française d’Endocrinologie (SFE).
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Disclose summary: The authors have nothing to disclose.
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Abbreviations: EB, embryoid bodies; ES, embryonic stem (cell); hMR, human mineralocorticoid
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receptor; GC, glucocorticoid; GR, glucocorticoid receptor; MAP2, microtubule associated protein 2;
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MR, mineralocorticoid receptor; PCNA, proliferating cell nuclear antigen; t-BHP, tert-
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butylhydroperoxide; WT, wild-type.
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Abstract
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Mineralocorticoid receptor (MR), highly expressed in the hippocampus, binds corticosteroid hormones
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and coordinately participates, with the glucocorticoid receptor (GR), to the control of stress responses,
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memorization and behavior. To investigate the impact of MR in neuronal survival, we generated
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murine embryonic stem (ES) cells that overexpress human MR (P1-hMR) and are induced to
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differentiate into mature neurons. We showed that recombinant MR expression increased throughout
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differentiation and is 2-fold higher in P1-hMR ES-derived neurons compared to wild type (WT)
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controls while GR expression was unaffected. Althought proliferation and early neuronal
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differentiation were comparable in P1-hMR and WT ES cells, MR overexpression was associated with
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higher late neuronal marker expression (MAP2, -tubulin III). This was accompanied by a shift
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towards neuron survival with an increased ratio of anti- vs pro-apoptotic molecules and 50% decreased
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caspase 3 activity. Knocking down MR overexpression by small interfering RNAs drastically reversed
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neuroprotective effects with reduced Bcl2/Bax ratio and decreased MAP2 expression. P1-hMR
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neurons were protected against oxidative stress-induced apoptosis through reduced caspase 3
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activation and drastically increased Bcl2/Bax ratio and -tubulin III expression. We demonstrated the
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involvement of MR in neuronal differentiation and survival and identify MR as an important
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neuroprotective mediator opening potential pharmacological strategies.
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Introduction
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The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, is highly expressed in
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the brain, notably in the hippocampus, where it is physiologically occupied and activated by
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glucocorticoid hormones (GC) (1). MR plays an important role in the neuroendocrine and behavioral
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responses to stress and in establishing cognitive functions (2). The classical nuclear MR is involved in
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the stability and integrity of neuronal networks (3). However, recent evidences suggest that rapid
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effects of GC depend on a membrane-located MR that modulates neuronal excitability (4-5). The
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central actions exerted by GC are also mediated by the lower affinity glucocorticoid receptor (GR).
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Thus, the MR/GR balance is of crucial importance to normalize brain activity and to regulate
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hippocampal plasticity (6).
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Several pharmacological studies and analyses of mouse models have shown that MR activation, in
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contrast to GR activation, is required for neuronal survival in the hippocampus (7-9). While
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stimulation of anti-apoptotic pathways by MR may partially explain its neuroprotective role (10-11), a
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rapid increase of MR expression following neuronal injury was reported (12), thus establishing a
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positive relationship between MR expression and neuroprotection. We have recently demonstrated that
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MR expression via transcriptional activation of its two promoters increase during neuronal
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differentiation of murine embryonic stem (ES) cells (13). However, the mechanisms by which MR
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promotes neuronal differentiation and maintains neuron survival remain unclear.
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The hippocampus is a major site of neurogenesis in adulthood. Specific MR activation enhances
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neonatal neurogenesis (14) thus promoting cognitive processes. Forebrain MR over-expression
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improves memory processes in mice (9), while MR knockout animals exhibit impaired learning
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abilities (15). Moreover, hippocampal neurons greatly decrease with age (16) in parallel with the
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hippocampal MR expression (17), indicating that reduced MR expression is associated with neuronal
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dysfunction in the hippocampus of older individuals.
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To investigate the impact of MR on neuronal survival and/or differentiation and better elucidate the
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molecular mechanisms involved, we exploited an ES cell model that could be committed to neuronal
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differentiation (13) and compared wild-type and hMR over-expressing ES cell lines derived from mice
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overexpressing hMR (18-19). These cell-based systems offer a unique opportunity to examine the
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functional consequences of MR over-expression on the regulation of the apoptosis signaling pathway
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during neuronal differentiation and in mature neurons. We showed that MR over-expression increases
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expression of the late neuronal markers that in turn, is associated with an increase in the ratio between
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anti- and pro-apoptotic molecules, providing direct evidence for an anti-apoptotic impact of neuronal
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MR.
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Materials and Methods
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Cell Culture
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A murine hMR-overexpressing ES cell line, in which the P1 promoter drives hMR cDNA expression,
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was derived as described (19). The wild-type D3 ES cell line (ATCC no. CRL-11693) and the hMR
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ES cells were grown on 0.1% gelatin-coated plates (Sigma-Aldrich, Lyon, France) and on feeder cells
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(STO Neomycin LIF, kindly provided by Dr Alan Bradley, The Wellcome Trust Sanger Institute, UK)
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pretreated with 15 µg/ml mitomycin C (Sigma-Aldrich) for 4 h. Cells were cultured at 37°C in a
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humidified incubator in presence of 5% CO2.
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Reagents - ES medium was composed of DMEM (PAA, Les Mureaux, France) containing 15% fetal
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calf serum (FCS specifically tested for ES culture (AbCys SA, Paris, France), 1X non-essential amino
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acids (PAA), 2 mM glutamine (PAA), 100 U/ml penicillin (PAA), 100 µg/ml streptomycin (PAA), 20
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mM HEPES (PAA) and 100 µM -mercaptoethanol (Sigma-Aldrich). Embryoid Bodies (EB) medium
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had a similar composition but contained 10% FCS without -mercaptoethanol. Cortisol and
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aldosterone concentrations in the serum batch used for all experiments were measured at 30.25 nM
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and 41 pM , respectively. Neuron medium was similar to EB medium but was supplemented with 5
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µg/ml insulin (Sigma-Aldrich), 5 µg/ml transferrin (Sigma-Aldrich), and 29 nM sodium selenate
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(Sigma-Aldrich).
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Differentiation of ES cells into Neuronal-like cells – Neuronal differentiation was induced in ES
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medium containing 15% FCS with retinoic acid (RA), as previously described, via embryoid bodies
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(EB) formation (13). Of note, we were unable to achieve neuronal differentiation of ES cells when
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cultivated during two weeks with medium containing Dextran-Charcoal Coated (DCC) serum.
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Briefly, ES cells formed EB when exposed to 10-6 M Retinoic acid (Sigma-Aldrich) for 5 days in non-
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adhesive bacterial dishes. At day 7, EB were dissociated and incubated in neuron medium until day 14
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in adherence in tissue culture dishes. Cells were washed in PBS and froze before RNA or protein
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extraction.
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Cell Treatment – For hormonal treatment, after 24 h incubation in DCC medium, aldosterone (Acros
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Organics, Halluin, France), or corticosterone (Sigma-Aldrich), and/or RU486 (Mifepristone) (Sigma-
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Aldrich) were added to the culture medium at day 13 of the neuronal differentiation. After 6 h, total
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RNA was extracted with Trizol and gene expression was measured by quantitative real-time PCR. For
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apoptosis induction, cells were treated at day 14 with 400 µM tert-butylhydroperoxide (t-BHP)
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(Sigma-Aldrich) for 3 h in neuron medium containing 10% FCS. Successively, proteins were extracted
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and quantified by Western blot.
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Flow cytometry
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Cells were fixed and permeabilized using the Foxp3 Staining Buffer Set (eBioscience). Cells were
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then stained with anti-Ki67 antibody or with isotype control (BD Bioscience) for 30 min on ice. Flow
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cytometry was performed with a FACSCanto cytometer (BD Biosciences) and data files were
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analyzed using FlowJo software (Tree Star Inc.).
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Quantitative Real Time PCR
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Gene expression was quantified by real time PCR. Total RNA was processed for real time PCR on an
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ABI 7300 Sequence Detector (Applied Biosystems, Courtaboeuf, France). Briefly, 1 µg of total RNA
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was treated using the DNase I Amplification Grade procedure (Invitrogen). RNA was then reverse-
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transcribed with 50 U MultiScribe reverse transcriptase (Applied Biosystems). After 10-fold dilution,
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1/20th of the reverse transcription reaction was used for PCR using the Fast SYBR® Green PCR
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master mix (Applied Biosystems). Final primer concentrations were 300 nM for each primer (see
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Supplemental Table 1 for sequences). Reaction parameters were 50 °C for 2 min followed by 40
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cycles at 95°C for 15 s, and 60 °C for 1 min. For standard preparation amplicons were purified from
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agarose gel and subcloned into pGEMT-easy plasmid (Promega), then sequenced to confirm the
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identity of each fragment. Standard curves were generated using serial dilutions of linearized standard
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plasmids, spanning 6 orders of magnitude and yielding correlation coefficients >0.98 and efficiencies
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of at least 0.95, in all experiments. Standard and sample values were determined in duplicate from
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independent experiments. Relative expression within a given sample was calculated as the ratio:
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attomol of specific gene/femtomol of 18S. Results are mean ± S.E.M and represent the relative
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expression compared with that obtained with control cells, which was arbitrary set at 1.
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Western blot
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Total protein extracts were prepared from ES cells and neuron cultures. Cells were lysed, in lysis
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buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 30 mM Na pyrophosphate, 50 mM Na
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fluoride, 1% Triton X100, 1X protease inhibitor from Sigma) on ice. Immunoblots were incubated 1 h
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at room temperature in 5% fat free milk-Tris buffer saline – 0.1% Tween 20 (TBS-T) before overnight
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incubation at 4°C with one of the following antibodies: rabbit anti-MR 39N (1/1,000), mouse anti--
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tubulin III TU-20 (1/1,000) (Millipore, Molsheim, France), rabbit anti-Bcl2 (1/500) (Ozyme, Saint-
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Quentin-en-Yvelines, France), mouse anti-PCNA (1/1,000) (Dako, Trappes, France), rabbit anti-
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caspase 3 (1/1,000) (Ozyme), rabbit anti-Bax (1/15,000) (Ozyme) and mouse anti-GR (clone FIGR,
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Millipore, 1/500). After extensive washing, membranes were incubated for 30 min at room
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temperature with peroxydase-conjugated goat anti-rabbit (1/15,000) or anti-mouse (1/15,000)
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secondary antibodies (Vector Laboratory, Burlingame, CA). After washing, the antigen-antibody
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complex was visualized by the ECL+ detection kit (GE Healthcare Europe, Orsay, France). For loading
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normalization, membranes were incubated with rabbit anti-GAPDH (1/5,000) (Sigma-Aldrich) or
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mouse anti--tubulin (1/10,000) (Sigma-Aldrich). Signal intensities were quantified with QuantityOne
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software (Bio-Rad, Marnes-la-Coquette, France). Alternatively, the Odyssey imaging system (LI-COR
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Biosciences, Bad Homburg, Germany) was used for quantification with IRDye© 800CW or 680LT
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near-infrared fluorescent secondary antibodies.
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Confocal Immunofluorescence Microscopy
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Cells grown on sterile coverslips were fixed with methanol for 10 min, rinsed with PBS-0.1% Tween
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20 and incubated with a PBS, 5% BSA, 0.1% casein block for 20 min followed by overnight
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incubation at 4°C with the anti-MR 39N polyclonal antibody (4 µg/ml) then with Alexa Fluor 555 goat
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anti-rabbit (1/1,000) (Molecular Probes) for 1 h at room temperature. The cells were next rinsed in
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PBS, and incubated with the anti--tubulin III TU-20 monoclonal antibody (1/100) (AbCys) for 2 h at
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room temperature followed by washing and incubation with Alexa Fluor 488 goat anti-mouse antibody
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(1/1,000) (Molecular Probe) for 1 h at room temperature. The coverslips were then mounted with
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Fluorescence Mounting Medium (Dako), before analysis and imaging by confocal fluorescence
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microscopy (Zeiss HAL confocal microscope).
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MR knockdown by siRNA
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Neurons were transiently transfected at day 11 with 100 nM siRNA (Invitrogen; see Supplemental
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Table 1 for sequences), using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM Reduced Serum
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Medium (Invitrogen) according to the manufacturer’s recommendations. Six hours post-transfection,
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cells were incubated in neuron medium for 48 h. At day 14, total RNA were extracted and gene
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expression was measured by qPCR.
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Statistical Analyses
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Results represent mean ± SEM of at least 6 samples for each condition unless stated otherwise.
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Statistical analyses were performed using a non parametric Mann-Whitney test (Prism4, Graphpad
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Software, Inc., San Diego, CA).
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Results
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MR over-expression during neuronal differentiation of ES cells
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The hMR over-expressing ES cell line was established from transgenic P1-hMR mouse blastocysts
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(19). The transgenic mice were generated using 1.2 kb of the human proximal MR promoter, named
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P1, to drive hMR cDNA expression (18). To investigate the impact of MR over-expression during
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neuronal differentiation, we first examined the expression of hMR transgene mRNA in the
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recombinant ES cells by quantitative real-time PCR and showed that hMR transcript levels rose
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approximately 3.5-fold in mature neurons compared to undifferentiated ES cells (Fig. 1A). We next
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analyzed MR protein expression during neuronal differentiation in transgenic ES cells (P1-hMR)
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compared with wild-type (WT) using an anti-MR antibody recognizing both the endogenous murine
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MR and recombinant human MR (20). Western blot analyses revealed an approximately 1.6-fold
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increase of MR expression in the P1-hMR ES cells compared to WT ES cells and 1.7-fold increase in
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the P1-hMR neurons compared to WT neurons (Fig. 1B). In parallel, we showed that while
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endogenous mMR mRNA expression remains identical in undifferentiated P1-hMR and WT ES cells,
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differentiated neurons of both genotypes under the same experimental conditions exhibit a 3-fold
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increase in mMR transcripts without significant difference between transgenic and WT ES cell lines
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(Fig. 1C). Similarly, the presence of the transgene did not modify the expression of the closely related
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glucocorticoid receptor (GR) both at the mRNA and protein levels as measured by real-time qPCR
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during neuronal differentiation and western blot at d14 (Fig. 1D and E). This indicated that hMR
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overexpression does not affect endogenous corticosteroid receptor abundance in mature neurons.
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Double-immunolabeling experiments with the anti-MR and anti--tubulin III antibodies clearly reveal
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a colocalization of MR and -tubulin III (Merge panel Fig. 1F) showing that MR is almost exclusively
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expressed in mature, -tubulin III-positive neurons. Altogether, these results demonstrate that ES cell-
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derived neurons provide an effective cell-based system to investigate the functional consequences of
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hMR over-expression during neuronal differentiation.
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Impact of MR over-expression on neuronal differentiation
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In order to examine the impact of MR over-expression, transgenic and WT ES cells were
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differentiated into neurons, and the variations of the expression levels of several specific neuronal
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markers were analyzed by quantitative real-time PCR. Under our experimental conditions where the
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ligand-dependent transcription factor MR was activated by corticosteroid hormones present in the
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serum containing medium, the expression profile of the neuronal progenitor marker nestin was similar
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in the ES cell lines of both genotypes during neuronal differentiation (Fig. 2A), suggesting that MR
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over-expression does not affect early neuronal commitment. Besides, the expression of the mature
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neuronal marker Microtubule-Associated Protein 2 (MAP2) was very low in undifferentiated ES cells
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but readily increased, as expected, in mature neurons. We performed neuronal differentiation of
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another WT ES cell line (19), assessing the expression of two late neuronal markers MAP2 and
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synaptophysin compared to the WT D3 ES cell line and did not found any significant difference (see
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supplemental Fig. S1). Interestingly, we showed that the MAP2 mRNA level was 4.5-fold higher in
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P1-hMR neurons than in WT controls (Fig. 2B). In addition, western blot analysis showed a 1.7-fold
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increase of another late neuronal marker -tubulin III in P1-hMR neurons compared to WT neurons
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(Fig. 2C). Several hypotheses could account for these observations: MR over-expression might
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facilitate the differentiation of precursors into neuronal lineage and could promote the growth of
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mature neurons. An alternative and not mutually exclusive hypothesis is that MR-over-expression is
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associated with an increased survival of newly differentiated neurons.
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MR over-expression favors the increased survival of neurons
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The increased expression of late neuronal markers reflects an increase of neuronal proliferation or
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survival. We thus examined by Western blot the expression of the proliferation marker, PCNA
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(Proliferating Cell Nuclear Antigen) in neurons and did not detect any significant difference between
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WT or P1-hMR neurons (Fig. 3A). This result was confirmed by Fluorescence Activated Cell Sorting
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method, using an anti-Ki67 (another proliferation marker) antibody, (56.3% Ki67 positive WT cells vs
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57.0% Ki67 positive P1.hMR cells at d13 of differentiation, see supplemental Fig. S2), thus indicating
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that MR over-expression has no major impact on neuron proliferation. We then examined by Western
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blot the cleavage of caspase 3, as an index of caspase 3 activation and an indirect marker of apoptosis,
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and showed a 57% reduction of caspase 3 cleavage in MR over-expressing neurons compared to WT
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(Fig. 3B), suggesting that MR over-expression may confer resistance to apoptotic cell death thus
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facilitating neuron survival.
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Functional consequences of MR over-expression on neuron survival
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To determine the impact of MR over-expression on neuron survival, we studied the expression of
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transcripts and proteins encoded by the Bcl2 gene family during neuronal differentiation of ES cell
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lines of both genotypes. The Bcl2 gene family is a major regulatory component of the apoptotic
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pathway comprising death inducers and death repressors. These proteins are activated by different
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stimuli and represent upstream events leading to the conclusive phase of the apoptotic process
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involving caspase 3 activation. The ratio between death inducers and repressors is a key element
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determining cell survival or death (21-22). Specifically we examined the expression of two anti-
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apoptotic markers: Bcl2 and BclxL, and two pro-apoptotic markers: Bax and Bak. Quantitative real-
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time PCR analysis indicated that the expression of Bcl2 transcripts increases 6.5- and 23.7-fold during
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neuronal differentiation of WT and P1-hMR ES cell lines, respectively (Fig. 4A), Bcl2 mRNA
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expression being significantly and reproducibly higher in P1-hMR than in WT neurons. Moreover, P1-
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hMR neurons exhibited a 2.5-fold rise of BclxL transcript levels compared to undifferentiated P1-hMR
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ES cells, whereas no statistical difference in BclxL expression was detected during neuronal
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differentiation of the WT ES cell line (Fig. 4B). Taken together, these findings strongly support a
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positive relationship between MR over-expression and an increase in the expression of anti-apoptotic
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markers. In contrast, steady state levels of Bax and Bak mRNA decreased by approximately 50% in
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WT ES cell-derived neurons but remained constant in P1-hMR neurons during neuronal differentiation
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(Figure 4C and 4D). Finally, of major interest, the ratios of both mRNA (Fig. 4E) and protein (Fig.
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4F) between anti-apoptotic and pro-apoptotic markers were always higher in P1-hMR that in WT
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neurons, providing strong evidence that MR over-expression promotes anti-apoptotic factors
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expression, thus facilitating neuronal survival.
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MR knockdown inhibits neuronal-specific increase in anti-apoptotic markers
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To confirm that MR over-expression enhances neuronal differentiation and stimulates neuronal
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survival, a small interfering RNA (siRNA) strategy was exploited using two unrelated MR specific
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siRNAs in P1-hMR ES-derived neurons. In Fig. 5 is illustrated the decrease of mMR and hMR mRNA
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expression (approximately 67 % and 57 %, respectively), obtained 48h post-transfection with the
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respective siRNA compared with scrambled siRNA (Fig. 5A-B). This reduction was accompanied not
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only by a significant and concomitant diminution of the mRNA levels of the late neuronal marker
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MAP2 (98% and 86 %) but also by a decrease of the anti-apoptotic marker Bcl2 (Fig. 5C-D). In
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parallel, the two MR siRNAs induced about a 50% increase of the relative expression of the pro-
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apoptotic marker Bax (Fig. 5E). Finally, of major interest, the two MR siRNAs caused a marked
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reduction of the anti-apoptotic to pro-apoptotic ratio (66%) (Fig. 5F). Collectively, these findings
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bring additional support for MR involvement in the increased expression of late neuronal markers. We
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also provide evidence that MR knock-down blunts the increase of anti-apoptotic markers associated
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with MR over-expression while facilitating the decrease of pro-apoptotic markers expression,
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validating the anti-apoptotic role of this receptor.
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The relative level of MR is crucial for the anti-apoptotic effect
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We decided then to investigate the impact of steroid hormones on the ratio of anti-apoptotic to pro-
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apoptotic markers, in ES-cell derived neurons. Cells were incubated with aldosterone or corticosterone
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at d13 of differentiation. Steroid-induced modification of Bcl2 and Bax mRNA expression was
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measured after 6 h treatment using quantitative real-time PCR. As shown in Fig. 6, 100 nM
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aldosterone had no consequence on the ratio of anti-apoptotic to pro-apoptotic marker on both
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genotypes. In sharp contrast, corticosterone had a differential effect on P1-hMR neurons compared to
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WT neurons. A 35% decrease of Bcl2 to Bax ratio was observed in WT neurons, whereas a 2.3-fold
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increase was observed in P1-hMR neurons. Corticosterone-induced effects were not affected by
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RU486, a GR antagonist, unambiguously demonstrating MR involvement in controlling the anti-
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apoptotic/pro-apoptotic signal balance. Collectively, these findings show that MR not only controls
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gene expression of death repressors and inducers but more importantly that neuronal MR abundance
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also dictates the extent and the direction towards pro- or anti-apoptotic phenotype.
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Neuronal MR over-expression reduces t-BHP-induced cell death
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To examine the effect of MR over-expression on oxidative stress-induced apoptosis, we compared the
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survival of WT and of MR over-expressing neurons after 3 h exposure to 400 µM tert-
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butylhydroperoxide (t-BHP). t-BHP treatment led to characteristic WT cell morphological changes
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including round shape of neurons, beading followed by extensive degeneration of the neurites and lost
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of neuronal integrity, many cells detaching from the culture plate. In contrast, under similar
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experimental conditions, P1-hMR neurons appeared almost normal with only few floating cells.
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Western blot analyses show that the t-BHP-induced caspase 3 cleavage is 3-fold higher in WT than in
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P1-hMR neurons (Fig. 7A). Likewise, the ratio between anti-apoptotic and pro-apoptotic markers is 5-
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fold higher in P1-hMR than in WT neurons (Fig. 7B). Additionally, exposure of cultures to t-BHP
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induces to a drastic reduction of -tubulin III protein expression in WT compared to P1-hMR neurons
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(Fig. 7C), supporting the morphological changes. Altogether, these data demonstrate that MR over-
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expression is associated with a significant protection against t-BHP-induced neuronal death.
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Discussion
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In this present work, we investigated whether and how MR controls neuronal differentiation and/or
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survival using a model of MR over-expression in ES cell-derived neurons obtained from P1-hMR
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transgenic mice (18-19). In this cell-based system, P1-hMR neurons exhibit a 2-fold increase in MR
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protein expression compared to differentiated WT neurons while GR expression level remains
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unchanged, leading to a moderately enhanced MR/GR ratio. To our knowledge, this is the first report
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that directly quantified the extent of neuronal MR overexpression at the protein level. This parameter
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is lacking in other brain-specific MR overexpression transgenic models (9, 23).
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Given that the relative receptor density and their occupancy by corticosteroid hormones are known to
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greatly affect neuronal maintenance, transmission and damage (2), our ES-derived neurons in which
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expression of one component of the corticosteroid signaling is specifically modified, constitute an
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appropriate experimental system. Even though one limitation of our model is that we could not
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directly control the concentration of corticosteroid hormones provided by the serum during the initial
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steps of neuronal differentiation, our cell based model remains suitable to clarify neuronal MR
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influence on cell differentiation, proliferation and susceptibility to cell apoptosis. Herein, we show that
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MR over-expression from early neuronal developmental stages and onwards is associated with
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increased expression of late neuronal differentiation markers. We unambiguously establish the pivotal
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role of MR in controlling the balance between anti- and pro-apoptotic signals as confirmed by
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knocking down MR expression with small interfering RNA strategy. We finally demonstrate the
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importance of MR abundance in conferring relative resistance to oxidative stress-induced cell
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apoptosis thus facilitating neuronal survival.
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MR and GR are abundantly expressed in neurons of the limbic areas where they mediate quite
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opposite effects. MR and GR exhibit distinct functional properties notwithstanding their similarities of
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structure, and mechanisms of action. Most notably both receptors bind glucocorticoid hormones
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(cortisol in humans, corticosterone in rodents) but GR presents a low affinity while MR has a 10 fold
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higher affinity for glucocorticoids (24). In addition, as ligand-dependent transcription factors, MR and
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GR recruit similar but also distinct coregulators which may partially account for the diversity of
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neuronal responses to glucocorticoids (25-27). Recent accumulating evidences show that acutely or
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chronically unbalanced glucocorticoid concentrations differentially affect neuronal function. Under
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rest conditions, basal levels of glucocorticoids which predominantly activated MR are essential for
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neuronal development, integrity and function. On the other hand, under stress exposure, high levels of
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glucocorticoids, which fully occupied and activated GR, are detrimental and induce neuronal death (8)
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though cell cycle arrest and activation of apoptosis signaling pathways (11, 28-29). Repeated stressful
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events trigger the damaging effects of GR on neurons and brain functions (6, 30). Thus, the
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coordinated activation of MR/GR pathways appears to be a major and critical regulator of neuronal
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function. Yet, the extent of MR signaling activation in the central nervous system seems to depend on
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the MR abundance beside corticosteroid hormone levels. In this respect, our model of MR over-
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expressing ES derived neurons conveys important new informations concerning MR influence in
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neuronal determination and survival.
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It is well established that the ratio between death repressors or anti-apoptotic molecules (e.g. Bcl2,
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BclxL) and death inducers (e.g. Bax, Bak) or pro-apoptotic markers is determinant for cell fate (21-22).
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Under basal conditions, Bcl2 and BclxL sequester by dimerization Bax and Bak in the cytosol, thus
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preventing their migration to mitochondria and apoptosis. However, when the amount of repressors is
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insufficient to neutralize all the pro-apoptotic molecules, apoptotic signals prevail leading to caspase
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activation and apoptosis (31-32).
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We demonstrated that the anti-apoptotic/pro-apoptotic molecule ratio is much higher in MR over-
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expressing neurons than in WT neurons, supporting a neuroprotective role of MR. As previously
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reported on other models (10-11), the involvement of neuronal MR in regulating apoptosis signaling
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pathways is corroborated by several lines of evidence.
360
First, we show that corticosterone, but not aldosterone exposure of WT neurons significantly increases
361
the pro-apoptotic potential while corticosterone exerts an anti-apoptotic effect on P1-hMR neurons.
362
These opposite actions of corticosterone persist in presence of the GR antagonist RU486, identifying
363
MR as a pro-survival factor and underlying the role of MR over-expression in conferring neuronal
364
resistance to apoptotic signals. These findings are in agreement with previous in vitro and in vivo
365
studies that reported a rapid upregulation of MR (mRNA and protein) associated with an increased
366
survival of rat primary cortical neurons in response to mild injury and in rat hippocampus following
367
hypothermic transient global ischemia (12). The in vivo neuroprotective effect of MR was further
368
demonstrated by transgenic mice presenting specific forebrain MR over-expression. These animals
369
exhibited a decreased sensitivity to stress, anxiety-like behavior and enhanced memory (9, 23). More
370
importantly, these transgenic mice presented with attenuated hippocampal neuron loss after cerebral
371
ischemia, consistent with the increased survival of MR over-expressing ES-derived neurons we
372
described.
373
Second, to validate the assumption the MR over-expression confers apoptosis resistance, we
374
performed MR knockdown in P1-hMR neurons by a siRNA strategy. Along with the marked
375
reduction of MR expression, a significant decrease of MAP2 expression was observed consistent with
376
a massive loss of mature neurons associated with a reduced anti/pro-apoptotic Bcl2/Bax ratio. Taken
377
together, there is a clear positive relationship between MR abundance, anti-/pro-apoptotic factor
16
378
expression ratio and neuronal marker level. This observation is in agreement with the forebrain
379
specific genetic disruption of MR in mice, which associates altered learning processes and dentate
380
granule cell degeneration (7, 15). Taken together, these findings provide strong evidence that
381
increased in vitro and in vivo MR expression is directly and causally linked to the promotion of
382
neuronal survival.
383
Third, additional results corroborate the prominent role of activated MR signaling in preventing
384
neuronal cell death-signaling cascade. We explored cell viability after acute exposure of neurons to t-
385
BHP, a strong inducer of oxidative stress. We show that MR overexpressing neurons were resistant to
386
oxidative injury as revealed by the reduction in caspase 3 cleavage and the sharp increase in b-tubulin
387
III protein expression monitoring neuronal survival. Interestingly, several physiological or
388
pathophysiological conditions are clearly associated with an increased expression of brain MR such as
389
during aging (17), after antidepressant imipramine treatment (33), in depressed patients (34) or after
390
cerebral ischemia (35).
391
The molecular mechanisms by which MR may regulate gene expression of the Bcl 2 family members
392
remain to be established. As a transcription factor, MR may directly or indirectly interact with the
393
regulatory sequences of anti-apoptotic genes to modulate their transcription. Several groups have
394
identified potential hormone responsive elements in BclxL and Bcl2 gene promoters which specifically
395
bind PR and GR in vitro and in vivo (36-38). Given that MR binds to the same consensus HRE
396
sequence, it is tempting to speculate that MR may regulate Bcl 2 and BclxL gene expression by acting
397
directly on the HRE sequences located at their promoters, in the context of neuronal survival in
398
rodents (39). This does not exclude that MR activates or represses other specific sets of target genes
399
essential for neuronal survival program.
400
We also surmise that the shift of the pro-apoptotic/anti-apoptotic balance towards neuron survival may
401
account for the higher expression of late neuronal markers in P1-hMR neurons. Indeed, it has been
402
previously proposed that anti-apoptotic factors facilitate neuronal differentiation, whereas a reduction
403
of pro-apoptotic factors expression was observed by several groups (40-44). Therefore, besides their
404
role in cell death, the proteins of Bcl2 family are largely implicated in neurogenesis.
17
405
An additional layer of complexity is given by the putative membrane-located MR which exerts rapid
406
non-genomic actions resulting in the stimulation of the frequency of excitatory postsynaptic glutamate
407
currents in the mouse hippocampus. This effect was blocked by MR specific antagonist
408
spironolactone, and did not occur in brain specific MR knockout mice (5). Surprisingly, this
409
membrane-located MR seems to have a 10 to 20 fold lower affinity for GC than the intracellular MR.
410
Interestingly, MR has been recently detected in the membranes of rat amygdala glutamatergic and
411
GABAergic neurons, with a presynaptic and postsynaptic localization (45). Whether this membrane
412
MR is involved in neuronal survival remains to be elucidated.
413
In conclusion, we have successfully established a novel model of MR over-expression using the
414
neuronal differentiation of ES cells that was proven to be a suitable cell-based system to investigate
415
many functions of neuronal MR. This alternative approach fully complementing previous cellular and
416
animals models should facilitate the development of therapeutic strategies designed to improve
417
neuronal MR signaling efficiency and thereby opening new means to prevent or attenuate neuronal
418
cell apoptosis in neurodegenerative diseases.
419
420
Acknowledgments:
421
We thank Federico Simonetta (UMR_S 1012, Le Kremlin Bicêtre, France) for his help with FACS
422
experiments.
423
18
424
References
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Reul JM, de Kloet ER 1985 Two receptor systems for corticosterone in rat brain:
microdistribution and differential occupation. Endocrinology 117:2505-2511
de Kloet ER, Joels M, Holsboer F 2005 Stress and the brain: from adaptation to disease. Nat
Rev Neuroscience 6:463-475
de Kloet ER, Karst H, Joels M 2008 Corticosteroid hormones in the central stress response:
quick-and-slow. Front Neuroendocrinol 29:268-272
Olijslagers JE, de Kloet ER, Elgersma Y, van Woerden GM, Joels M, Karst H 2008 Rapid
changes in hippocampal CA1 pyramidal cell function via pre- as well as postsynaptic
membrane mineralocorticoid receptors. Eur J Neurosci 27:2542-2550
Karst H, Berger S, Turiault M, Tronche F, Schutz G, Joels M 2005 Mineralocorticoid receptors
are indispensable for nongenomic modulation of hippocampal glutamate transmission by
corticosterone. Proc Natl Acad Sci U S A 102:19204-19207
Joels M, Karst H, DeRijk R, de Kloet ER 2008 The coming out of the brain mineralocorticoid
receptor. Trends Neurosci 31:1-7
Gass P, Kretz O, Wolfer DP, Berger S, Tronche F, Reichardt HM, Kellendonk C, Lipp HP,
Schmid W, Schutz G 2000 Genetic disruption of mineralocorticoid receptor leads to impaired
neurogenesis and granule cell degeneration in the hippocampus of adult mice. EMBO Rep
1:447-451
Crochemore C, Lu J, Wu Y, Liposits Z, Sousa N, Holsboer F, Almeida OF 2005 Direct targeting
of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid
receptor activation. Mol Psychiatry 10:790-798
Lai M, Horsburgh K, Bae SE, Carter RN, Stenvers DJ, Fowler JH, Yau JL, Gomez-Sanchez CE,
Holmes MC, Kenyon CJ, Seckl JR, Macleod MR 2007 Forebrain mineralocorticoid receptor
overexpression enhances memory, reduces anxiety and attenuates neuronal loss in cerebral
ischaemia. Eur J Neurosci 25:1832-1842
McCullers DL, Herman JP 1998 Mineralocorticoid receptors regulate bcl-2 and p53 mRNA
expression in hippocampus. Neuroreport 9:3085-3089
Almeida OF, Conde GL, Crochemore C, Demeneix BA, Fischer D, Hassan AH, Meyer M,
Holsboer F, Michaelidis TM 2000 Subtle shifts in the ratio between pro- and antiapoptotic
molecules after activation of corticosteroid receptors decide neuronal fate. Faseb J 14:779790
Macleod MR, Johansson IM, Soderstrom I, Lai M, Gido G, Wieloch T, Seckl JR, Olsson T 2003
Mineralocorticoid receptor expression and increased survival following neuronal injury. Eur J
Neurosci 17:1549-1555
Munier M, Meduri G, Viengchareun S, Leclerc P, Le Menuet D, Lombes M 2010 Regulation
of mineralocorticoid receptor expression during neuronal differentiation of murine
embryonic stem cells. Endocrinology 151:2244-2254
Fujioka A, Fujioka T, Ishida Y, Maekawa T, Nakamura S 2006 Differential effects of prenatal
stress on the morphological maturation of hippocampal neurons. Neuroscience 141:907-915
Berger S, Wolfer DP, Selbach O, Alter H, Erdmann G, Reichardt HM, Chepkova AN, Welzl H,
Haas HL, Lipp HP, Schutz G 2006 Loss of the limbic mineralocorticoid receptor impairs
behavioral plasticity. Proc Natl Acad Sci U S A 103:195-200
De Nicola AF, Pietranera L, Beauquis J, Ferrini MG, Saravia FE 2009 Steroid protection in
aging and age-associated diseases. Exp Gerontol 44:34-40
Choi JH, Hwang IK, Lee CH, Chung DW, Yoo KY, Li H, Won MH, Seong JK, Yoon YS, Lee IS
2008 Immunoreactivities and levels of mineralocorticoid and glucocorticoid receptors in the
hippocampal CA1 region and dentate gyrus of adult and aged dogs. Neurochem Res 33:562568
19
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
Le Menuet D, Isnard R, Bichara M, Viengchareun S, Muffat-Joly M, Walker F, Zennaro MC,
Lombes M 2001 Alteration of cardiac and renal functions in transgenic mice overexpressing
human mineralocorticoid receptor. J Biol Chem 276:38911-38920.
Le Menuet D, Munier M, Meduri G, Viengchareun S, Lombes M 2010 Mineralocorticoid
receptor overexpression in embryonic stem cell-derived cardiomyocytes increases their
beating frequency. Cardiovasc Res 87:467-475
Viengchareun S, Kamenicky P, Teixeira M, Butlen D, Meduri G, Blanchard-Gutton N,
Kurschat C, Lanel A, Martinerie L, Sztal-Mazer S, Blot-Chabaud M, Ferrary E, Cherradi N,
Lombes M 2009 Osmotic stress regulates mineralocorticoid receptor expression in a novel
aldosterone-sensitive cortical collecting duct cell line. Mol Endocrinol 23:1948-1962
Youle RJ, Strasser A 2008 The BCL-2 protein family: opposing activities that mediate cell
death. Nat Rev Mol Cell Biol 9:47-59
Hotchkiss RS, Strasser A, McDunn JE, Swanson PE 2009 Cell death. N Engl J Med 361:15701583
Rozeboom AM, Akil H, Seasholtz AF 2007 Mineralocorticoid receptor overexpression in
forebrain decreases anxiety-like behavior and alters the stress response in mice. Proc Natl
Acad Sci U S A 104:4688-4693
Reagan LP, McEwen BS 1997 Controversies surrounding glucocorticoid-mediated cell death
in the hippocampus. J Chem Neuroanat 13:149-167
Obradovic D, Tirard M, Nemethy Z, Hirsch O, Gronemeyer H, Almeida OF 2004 DAXX,
FLASH, and FAF-1 modulate mineralocorticoid and glucocorticoid receptor-mediated
transcription in hippocampal cells--toward a basis for the opposite actions elicited by two
nuclear receptors? Mol Pharmacol 65:761-769
Tirard M, Jasbinsek J, Almeida OF, Michaelidis TM 2004 The manifold actions of the protein
inhibitor of activated STAT proteins on the transcriptional activity of mineralocorticoid and
glucocorticoid receptors in neural cells. J Mol Endocrinol 32:825-841
Pascual-Le Tallec L, Lombes M 2005 The mineralocorticoid receptor: a journey exploring its
diversity and specificity of action. Mol Endocrinol 19:2211-2221
Crochemore C, Michaelidis TM, Fischer D, Loeffler JP, Almeida OF 2002 Enhancement of p53
activity and inhibition of neural cell proliferation by glucocorticoid receptor activation. FASEB
J 16:761-770
Sundberg M, Savola S, Hienola A, Korhonen L, Lindholm D 2006 Glucocorticoid hormones
decrease proliferation of embryonic neural stem cells through ubiquitin-mediated
degradation of cyclin D1. J Neurosci 26:5402-5410
Joels M 2008 Functional actions of corticosteroids in the hippocampus. Eur J Pharmacol
583:312-321
Merry DE, Korsmeyer SJ 1997 Bcl-2 gene family in the nervous system. Annu Rev Neurosci
20:245-267
Adams JM, Cory S 1998 The Bcl-2 protein family: arbiters of cell survival. Science 281:13221326
Brady LS, Whitfield HJ, Jr., Fox RJ, Gold PW, Herkenham M 1991 Long-term antidepressant
administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and
mineralocorticoid receptor gene expression in rat brain. Therapeutic implications. J Clin
Invest 87:831-837
Wang SS, Kamphuis W, Huitinga I, Zhou JN, Swaab DF 2008 Gene expression analysis in the
human hypothalamus in depression by laser microdissection and real-time PCR: the presence
of multiple receptor imbalances. Mol Psychiatry 13:786-799, 741
Lai M, Bae SE, Bell JE, Seckl JR, Macleod MR 2009 Mineralocorticoid receptor mRNA
expression is increased in human hippocampus following brief cerebral ischaemia.
Neuropathol Appl Neurobiol 35:156-164
Viegas LR, Vicent GP, Baranao JL, Beato M, Pecci A 2004 Steroid hormones induce bcl-X
gene expression through direct activation of distal promoter P4. J Biol Chem 279:9831-9839
20
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
37.
38.
39.
40.
41.
42.
43.
44.
45.
Gascoyne DM, Kypta RM, Vivanco MM 2003 Glucocorticoids inhibit apoptosis during
fibrosarcoma development by transcriptionally activating Bcl-xL. J Biol Chem 278:1802218029
Yin P, Lin Z, Cheng YH, Marsh EE, Utsunomiya H, Ishikawa H, Xue Q, Reierstad S, Innes J,
Thung S, Kim JJ, Xu E, Bulun SE 2007 Progesterone receptor regulates Bcl-2 gene expression
through direct binding to its promoter region in uterine leiomyoma cells. J Clin Endocrinol
Metab 92:4459-4466
Balsamo A, Cicognani A, Gennari M, Sippell WG, Menabo S, Baronio F, Riepe FG 2007
Functional characterization of naturally occurring NR3C2 gene mutations in Italian patients
suffering from pseudohypoaldosteronism type 1. Eur J Endocrinol 156:249-256
Trouillas M, Saucourt C, Duval D, Gauthereau X, Thibault C, Dembele D, Feraud O, Menager
J, Rallu M, Pradier L, Boeuf H 2008 Bcl2, a transcriptional target of p38alpha, is critical for
neuronal commitment of mouse embryonic stem cells. Cell Death Differ 15:1450-1459
Liste I, Garcia-Garcia E, Martinez-Serrano A 2004 The generation of dopaminergic neurons
by human neural stem cells is enhanced by Bcl-XL, both in vitro and in vivo. J Neurosci
24:10786-10795
Liste I, Garcia-Garcia E, Bueno C, Martinez-Serrano A 2007 Bcl-XL modulates the
differentiation of immortalized human neural stem cells. Cell Death Differ 14:1880-1892
Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S, et
al. 1995 Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient
mice. Science 267:1506-1510
Courtois ET, Castillo CG, Seiz EG, Ramos M, Bueno C, Liste I, Martinez-Serrano A 2010 In
vitro and in vivo enhanced generation of human A9 dopamine neurons from neural stem
cells by Bcl-XL. J Biol Chem 285:9881-9897
Prager EM, Brielmaier J, Bergstrom HC, McGuire J, Johnson LR 2010 Localization of
mineralocorticoid receptors at mammalian synapses. PLoS One 5:e14344
553
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Figures and Legends
555
556
Figure 1: MR over-expression during neuronal differentiation
557
A) Relative hMR mRNA expression levels were determined using qPCR in undifferentiated ES cells
558
and neurons. Results are means ± SEM of two independent experiments of six samples performed in
559
duplicate for each developmental stage indicating the relative expression compared with basal levels
560
of ES (arbitrarily set at 1). ** P<0.01. Mann Whitney test. Relative mRNA expression is normalized to
561
18S rRNA expression (see Materials and Methods section). B) Western blot analyses of MR protein
562
expression in WT and P1-hMR ES cell lines. Undifferentiated ES and neurons lysates from each ES
563
cell line were processed for immunoblotting with anti-MR antibody. GAPDH was used as loading
564
control. MR was normalized to GAPDH protein levels after digitalization on a gel scanner with
565
QuantityOne software (Bio-Rad, Marnes-la-Coquette, France). Results are presented as MR/GAPDH
566
ratio and as compared with basal levels of WT ES (arbitrarily set at 1). C-D) Relative mMR and mGR
567
mRNA expression levels were determined using qPCR in undifferentiated ES cells and neurons from
568
WT and P1-hMR ES cell lines. Results are means ± SEM of two independent experiments on six
569
samples performed in duplicate for each developmental stage and represent the relative expression
570
compared with basal levels of ES (arbitrarily set at 1). Mann Whitney test. Relative mRNA expression
571
is normalized to 18S rRNA expression (see Materials and Methods section). E) Western blot analysis
572
of GR expression in WT and P1.hMR neurons and signal quantification of the GR/GAPDH ratio (n =
573
6), ns: non significant. WT mean value arbitrarily set at 1. F) Double-immunolabeling of P1-hMR
574
neurons with antibodies against β-tubulin III (green) (left panel) and MR (red) (middle panel); merged
575
images are shown on the right. Original magnification x 40.
576
577
Figure 2: MR over-expression stimulates late neuronal markers without increasing neuronal
578
proliferation
579
A-B) Relative nestin and MAP2 mRNA expression levels were determined using qPCR in
580
undifferentiated ES cells and neurons. Results are means ± SEM of two independent experiments of
581
six samples performed in duplicate for each developmental stage and represent the relative expression
22
582
compared with levels of WT. P1-hMR and WT undifferentiated ES cell set arbitrarily at 1. ***
583
P<0.001 Mann Whitney test. Relative mRNA expression is normalized to 18S rRNA expression (see
584
Materials and Methods section). C) Western blot analyses of β-tubulin III protein expression in WT
585
and P1-hMR ES cell lines. Undifferentiated ES and neurons lysates from each ES cell line were
586
processed for immunoblotting with anti-β-tubulin III antibody. GAPDH was used as loading control.
587
b-tubulin III levels were normalized to GAPDH protein levels after digitalization on a gel scanner with
588
QuantityOne software (Bio-Rad, Marnes-la-Coquette, France). Results are presented as β-tubulin III
589
/GAPDH ratio and as compared with basal levels of WT neurons (arbitrarily set at 1).
590
591
Figure 3: MR over-expression does not affect cell proliferation but reduces Caspase 3 activity in
592
neurons
593
A) Western blot analyses of PCNA protein expression in WT and P1-hMR neurons. Neurons lysates
594
from each ES cell line were processed for immunoblotting with anti-PCNA antibody. GAPDH was
595
used as loading control. PCNA was normalized to GAPDH protein levels after digitalization on a gel
596
scanner with QuantityOne software (Bio-Rad, Marnes-la-Coquette, France). Results are presented as
597
ratio PCNA/GAPDH and as compared with basal levels of WT neurons (arbitrarily set at 1). B)
598
Caspase 3 activity was analyzed by western blot expression in WT and P1-hMR neurons. Lysates were
599
processed for immunoblotting with an antibody recognizing both caspase 3 and cleaved-caspase 3.
600
Protein levels were quantifued after digitalization on a gel scanner using QuantityOne software (Bio-
601
Rad, Marnes-la-Coquette, France). Results are presented as ratio cleaved-caspase 3 /caspase 3 of 6
602
samples and as compared with basal levels of WT neurons (arbitrarily set at 1). ** P<0.01. Mann
603
Whitney test.
604
605
Figure 4: Anti-apoptotic factors expression is increased in P1-hMR neurons
606
A-B-C-D) Relative Bcl2, BclxL, Bax, and Bak mRNA expression levels were determined using qPCR
607
in undifferentiated ES cells and neurons. Results are means ± SEM of two independent experiments of
608
six samples performed in duplicate for each developmental stage and represent the relative expression
609
compared with levels of undifferentiated ES cell set arbitrarily at 1. *** P<0.001 Mann Whitney test.
23
610
Relative mRNA expression is normalized to 18S rRNA expression (see Materials and Methods
611
section). E) The table represents the ratio between the mean of each anti-apoptotic marker expression
612
to the mean of each pro-apoptotic marker expression in neuronal state. F) Western blot analyses of
613
Bcl2 and Bax protein expression in WT and P1-hMR neurons. Lysates were processed for
614
immunoblotting with anti-Bcl2 or Bax antibody. GAPDH was used as loading control. Bcl2 and Bax
615
were normalized to GAPDH protein levels after digitalization on a gel scanner by using QuantityOne
616
software (Bio-Rad, Marnes-la-Coquette, France). The table represents the ratio between Bcl2 to Bax
617
expression in neuronal state.
618
619
Figure 5: MR down-regulation inhibits MR-mediated neuroprotective effects
620
P1-hMR neurons were transfected with either the control scrambled siRNA (scr MR) or by two
621
unrelated MR siRNA (si1 MR, si2 MR). A-E) Relative mMR, hMR, MAP2, Bcl2, Bax mRNA
622
expression levels were determined using qPCR. Results are means ± SEM of six samples performed in
623
duplicate and represent the relative expression compared with basal levels of control scrambled siRNA
624
transfected neurons (scr MR). **P<0.01, ***P<0.001. Mann Whitney test. Relative mRNA expression
625
is normalized to 18S rRNA expression (see Materials and Methods section). F) The graph represents
626
the ratio between the mean of anti-apoptotic marker Bcl2 expression to the mean of pro-apoptotic
627
marker Bax expression in each experimental condition.
628
629
Figure 6: Opposite effects of corticosterone-activated MR signaling on Bcl2/Bax ratio in WT and
630
P1-hMR neurons.
631
WT and P1-hMR neurons were exposed to 100 nM aldosterone (ALDO) and corticosterone (CORT)
632
in the absence or presence of 1 µM RU486. A-B) Results are means ± SEM (six samples performed in
633
duplicate), of ratio between the mean of anti-apoptotic marker Bcl2 expression to the mean of pro-
634
apoptotic marker Bax expression in neurons. ** P<0.01, * P<0.05. Mann Whitney test.
635
636
Figure 7: Neuronal MR over-expression confers resistance to oxidative stress-induced cell death
24
637
WT and P1-hMR neurons were exposed to 400 µM t-BHP for 4 hours. A) Caspase 3 activity was
638
analyzed by western blot expression in WT and P1-hMR neurons. Lysates were processed for
639
immunoblotting with antibody recognizing both caspase 3 and cleaved-caspase 3. Protein levels after
640
digitalization on a gel scanner by use QuantityOne software (Bio-Rad, Marnes-la-Coquette, France).
641
Results are presented as cleaved-caspase 3 /caspase 3 ratio (n = 9) and compared to ratio detected in
642
WT neurons (arbitrarily set at 1). * P<0.05. Mann Whitney test. B) Western blot analyses of Bcl2 and
643
Bax protein expression in WT and P1-hMR neurons. Neuron lysates from each ES cell line were
644
processed for immunoblotting with anti-Bcl2 or anti-Bax antibody. Results are presented as ratio Bcl2
645
/Bax (n=9) and compared with basal levels of WT neurons (arbitrarily set at 1). *** P<0.001. Mann
646
Whitney test.
647
C) Western blot analyses of β-tubulin III protein expression in WT and P1-hMR neurons. Neurons
648
lysates from each ES cell line were processed for immunoblotting with anti- β-tubulin III antibody.
649
GAPDH was used as loading control. -tubulin III was normalized to GAPDH protein levels after
650
digitalization on a gel scanner by use QuantityOne software (Bio-Rad, Marnes-la-Coquette, France).
651
Results are presented as ratio β-tubulin III /GAPDH (n = 6, **P<0.01. Mann Whitney test) and as
652
compared with basal levels of WT neurons (arbitrarily set at 1).
25